Sodium dodecyl sulfate – polyacrylamide gel electrophoresis

1. SDS- PAGE
Sodium dodecyl sulfate – polyacrylamide gel electrophoresis
.
Niharanjan PK.
DVM, Bangladesh Agricultural
University.

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Heat
Proteins are prepared by boiling in the
presence of SDS
SDS binding imparts a
constant charge/mass ratio
to the protein
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SDS
Sodium dodecyl sulfate

3. Running buffer: Tris-glycine-SDS, pH 8.3
Protein samples are
loaded into wells
Bromophenol blue in
sample buffer will act as
a tracking dye
At the start:
pH 6.8
pH 8.8

4. ____
+
Chloride ions in the stacking
gel buffer move rapidly to
positive pole
Proteins are negatively
charged and run at various
rates through the stacking gel
Glycine moves more slowly
than the proteins, since very
few glycine molecules have a
negative charge
Application Voltage
Glycine molecules enter stacking gel

5. Proteins "stack up" at the
interface between the two
gels
pH 8.8
+
Differential migration of
chloride and glycine ions sets
up a potential difference that
helps to concentrate proteins

6. +
Glycine (now negatively
charged) moves more rapidly
than proteins at the pH of
the running gel
Proteins are resolved by size
in the running gel
Migration of proteins (invisible) in
running gel is inversely proportional to
their log(MW)
Glycine amino groups lose a
proton as they enter the
running gel

7. Procedure
• Gather combs, glass plates, spacer (silicone tubing), and binder clip..A
comb is used to make wells (lanes) to load samples. Use an appropriate
comb depending on the sample size.
Example: Use an 8-lane comb for 7 samples and molecular weight
markers

8. • White petroleum jelly or 2% agar is applied around the edged
of the spacer to hold them in place and seal the chamber
between the glass plates
• Pour acrylamide solution for a separating gel. Overlay with
water to prevent contact with air (oxygen), which inhibits
polymerization. Allow acrylamide to polymerize for 20-30
minutes to form a gel.

9. • Remove water from the top gel and wash with stacking solution.
Pour stacking gel mixture, place the gel and allow to set
• After polymerization, comb is removed carefully and install the gel in
the electrophoresis apparatus
• Sample preparation: Add sample buffer(SDS) to samples, and mix
by flicking the tube. Sample heated in boiling water bath for 2-3min
and cool to room temperature for complete denaturation and
concentration of protein
• Take required volume of sample and inject into sample well using
micropipette
• Also load marker sample into few well
• Turn on the current to 10-15mA initially and continue to run at 30mA
through separating gel until sample reach the bottom (3/4) of the gel

10. • After the run is complete carefully remove the gel from
between the two plates and immerse in staining solution for at
least 3hours or overnight with uniform stacking
• Protein absorbs coomasive brilliant blue
• Transfer the gel to suitable container with at least 200-300ml
distaining solution and shake gently and continuously until the
gel background is colourless (unbound dye is removed)
• The protein fractionated into band are seen blue colour
• The gel can be now photographed or stored in polythene bags
and dried in vacuum for permanent record

11. Gels must be stained to visualize proteins
SDS-PAGE gels analyzed

12. Proteins are visualized with Coomassie Brilliant Blue G-250
Gels are rinsed with water several times to remove gel
chemicals and are then incubated with Simply Blue® a
colloidal suspension of Coomassie Blue G-250

13. BSA – 78K
CA – 45.7K
SBTI -
32.5K
B-gal 132K
Log10molecular
weight
Distance migrated (mm)
Sizes of proteins can be calculated by comparing their
migration to those of marker proteins